Background: Little is known about the association between apoC-III and lipoprotein-lipids in African American (AA) and Caucasian (CA) youths. The aim of this study was to investigate if plasma apoC-III level is associated with ethnicity differences in atherogenic lipoprotein-lipids between AA and CA youths.

High triglyceride (TG) levels coupled with low high-density lipoprotein cholesterol (HDLC) levels in circulating plasma are the hallmarks of hypertriglyceridemia[1] as well as a major risk factor for diabetes and cardiovascular diseases.[2-3] It is well known that African American (AA) adults and children have higher rates of obesity, diabetes and hypertension than Caucasian (CA) adults and children.[4] Paradoxically, AA adults and children have a lower prevalence of metabolic syndrome due to lower TG and higher HDLC levels,[4] as well as larger low-density lipoprotein cholesterol (LDLC)-particle sizes.[5-6] However, the mechanism(s) responsible for the ethnicity differences in atherogenic lipoprotein-lipid profiles are not clear, especially in youths.

One explanation for the ethnic differences in atherogenic lipoprotein-lipid profiles may be that AA children have lower visceral adiposity than CA children.[7] In a Bogalusa study, Freedman et al[8] found that the relationship of waist circumference with TG and very low-density lipoprotein (VLDL) subclasses was much greater in white youths than in black youths, suggesting that the magnitude of the deleterious effect of abdominal adiposity differs between the two ethnic groups. Similarly, Burns et al[5] found that lower visceral adiposity partially explained the ethnic differences in lipoprotein profiles in that black children had lower TG and VLDL concentrations and larger LDL particle sizes than white children.

Apolipoprotein C-III (apoC-III) plays a critical role in TG metabolism and therefore the TG concentrations in circulating plasma. ApoC-III consisting of 79-amino acids (8.8 kD) is synthesized predominantly in the liver and to a lesser extent in the intestine.[9] ApoC-III is a major component of VLDL and chylomicrons and a minor component of HDL.[9]

In the Monitored Atherosclerosis Regression Study, the amount of apoC-III in VLDL was found to be an independent predictor of the progression or severity of coronary artery disease.[10] In the Etude cas-t¨¦moin sur l' infarctus du myocarde study, elevated apoC-III levels were associated with myocardial infarction in non-diabetic subjects.[11] In young adults, increased apoC-III was an independent predictor of a family history of coronary artery disease that remained significant even after adjustment of apoB and TG levels.[12] However, it is not known if ethnic differences in apoC-III levels explain the ethnic differences in both lipoprotein-lipid profiles and their relationship with body fat, including abdominal differences between AA and CA youths.

The goals of the current study were: (i) to determine if plasma apoC-III levels differ between AA and CA youths; and (ii) to investigate how body fatness and markers of insulin resistance are associated with plasma apoC-III levels and atherogenic lipoprotein-lipids in AA and CA youths.

Methods

Subjects

In a cross-sectional design, a total of 202 children (mean age: 16.1¡À1.3 y, range: 13.8-18.9 y) consisting of 122 AA (boys/girls, 52/70) and 80 CA (boys/girls, 40/40) youths were recruited via flyers sent to local high schools. For AA youths, body mass index (BMI) values were 22.5¡À5.0 kg/m2 (range: 16.8-40.7) and 25.0¡À6.8 kg/m2 (range: 16.6-43.8) for boys and girls, respectively. For CA youths, BMI values were 22.0¡À4.8 kg/m2 (range: 17.1 -38.1) and 22.1¡À5.0 kg/m2 (range: 16.7- 45.5) for boys and girls, respectively. Interested youths and parents signed consent forms in accordance with the procedures of the Institutional Human Subject Committee.

Weight was assessed with a balance scale, and height was assessed using a stadiometer. These values were then used to calculate BMI (kg/m2). Waist and hip circumferences were measured at the level of the umbilicus and at the maximum posterior extension of the buttocks, respectively, with a commercially-available tape measure (MyoTape, AccuFitness, CO, USA), and waist-to-hip ratio (WHR) was calculated. A trained expert measured all body composition variables in triplicate, and the mean values were used. Total body composition was measured by dual energy X-ray absorptiometry (DXA) using the Hologic QDR-4500W (Waltham, MA, USA).

Plasma lipid, lipoprotein, and apolipoprotein measurements

A 20 mL fasting blood sample was obtained from an antecubital vein into Vacutainers® containing EDTA. Samples were separated by low speed centrifugation (1400 rpm) and stored at -80ºC. Plasma concentrations of total cholesterol (TC), LDLC, HDLC, TG, and glucose were determined by methods previously described.[13] Fasting insulin concentrations were also measured in duplicate using a commercially available ELISA immunoassay kit (ALPCO Diagnostics, Salem, NH, USA). The index of insulin resistance was assessed using the homoeostasis model of assessment-insulin resistance (HOMA-IR), as HOMA-IR = [fasting insulin (¦ÌIU/mL) ¡Á fasting glucose (mmol)]/22.5.[14] The coefficients of variations (CVs) for intra- and inter-assays of insulin were 4.3% and 6.8%, respectively.

Apolipoprotein A1, B and E were measured in duplicate using commercially available nephelometric immunoassays (Dade Behring, Marburg, Germany). The inter-assay CV for apoA-I, apoB and apoE was 4.7%, 6.6%, and 6.6%, respectively, and the intra-assay CV for apoA-I, apoB and apoE was 2.8%, 3%, and 4.4%, respectively. Serum apoC-III levels were measured in duplicate using a turbid metric immunoassay kit (Wako APO C3-HA, Wako Pure Chemical Inc., Osaka, Japan). The inter- and intra-assay variability for the assay was less than 10%.

LDL diameters were determined in duplicate using segmental gradient polyacrylamide gel (S-GGE 2.8/8.30) as previously described.[15] Briefly, plasma samples were pre-stained for 2 hours at room temperature with 0.6% Sudan black in ethylene glycol (3:1) prior to loading onto the gel. A single load of 10 ¦ÌL of prestained plasma solution was applied to individual troughs (GA-50 sample applicator, Isolabs). Electrophoresis was performed at room temperature for 18-20 hours (50 mA and 80 V). The position of the LDL bands was determined using a LKB laser densitometer (LKB Pharmacia, Piscataway, NJ, USA). The inter-and intra-assay CVs for the LDL particle diameter were 1.16% and 0.47%, respectively.

Statistical analyses

SPSS-PC version 10.1 was used for all statistical analyses. The distributions of the dependent variables were checked for normality prior to the analyses by plotting standardized residuals for each variable from regression models using ethnicity and gender as independent variables. Because BMI and waist circumference (WC) were not normally distributed, a log10 transformation was conducted, which adequately normalized the distributions of these variables.

Analyses of variance were used to compare gender and ethnic groups. Pearson correlations were calculated among body composition parameters, fasting insulin and HOMA-IR, apolipoproteins, and lipoprotein-lipids. Linear regression analyses were used to determine the degree to which gender, ethnicity, adiposity, and HOMA-IR explained the variance in apoC-III and other primary lipid variables. We chose WC as the primary index of central adiposity in the regression analyses for several reasons. Specifically, there were substantial inter-correlations among the adiposity indices; thus, we chose one index to avoid problems of colinearity. WC was more closely related to several measured lipoprotein-lipids including apoC-III than other adiposity indices (Table 2). Moreover, WC has consistently shown to be strongly associated with obesity-related metabolic disorders in adults[1] as well as in children,[8] perhaps because it focuses on abdominal adipose tissue. Finally, the use of WC avoided any problems associated with the use of ratios such as body fat percentage and BMI.[16]

Results

Table 1 shows the characteristics of the subjects. Ages were similar between AA and CA youths and between boys and girls. Girls had higher body fat than boys, while boys had higher WHR than girls, suggesting that girls have higher overall body fatness than boys, while boys are more centrally obese than girls. AA youths had significantly higher values in body weight and BMI values than CA youths. A further analysis showed that these differences in body composition were limited to the girl sample since AA girls had significantly higher body weight and BMI values than CA girls, with no such differences between AA and CA boys. The ethnicity differences in body weight and BMI may imply that AA girls have higher lean body mass including muscle mass and/or bone mineral density than CA girls. No significant differences in percent body fat, WHR, and WC were found between the two ethnicity groups.

With respect to lipoprotein-lipids and apolipoproteins, girls had generally more favorable lipoprotein-lipids profiles than boys, especially in HDLC and apoA-I. AA youths had more favorable lipoprotein-lipids featuring of low TG, low TC/HDLC, and high HDLC than CA youths. In particular, AA youths had lower apoC-III levels along with higher apoE levels than CA youths, while the former had higher fasting insulin and higher HOMA-IR than the latter. Boys had higher fasting glucose levels than girls (Table 1).

Tables 2 and 3 represent Pearson correlations among the measured variables by ethnicity. In CA youths, most of body composition parameters were positively associated with apoC-III, TG, and TC/HDLC and negatively associated with HDLC. In AA youths, WC, BMI, and WHR were positively associated with TG and/or TC/HDLC and negatively associated with HDLC. Regardless of ethnicity, fasting insulin and HOMA-IR were positively associated with TG and TC/HDLC. In CA, not AA, youths, insulin and HOMA-IR were also positively associated with circulating apoC-III and apoE.

Table 4 shows the final regression models for apoC-III and other major lipid variables for both CA and AA youths, respectively. WC and HOMA-IR were significant predictors for apoC-III in CA youths, but not in AA youths. HOMA-IR was a significant predictor for TG in both CA and AA youths. WC was a predictor for both HDLC and TC/HDLC in CA and AA youths.

Discussion

The findings of the present study are in line with those of previous studies, i.e., AA youths have more favorable lipoprotein-lipids than CA youths.[4-5,7] To our knowledge, this is the first article to report that AA youths have significantly lower apoC-III and higher apoE levels in circulating plasma even with higher fasting insulin and HOMA-IR than CA youths. Low apoC-III levels along with high apoE appear to be associated with the more favorable lipoprotein-lipids featuring of lower values in TG and TC/HDLC and higher values in HDLC and LDL particle size in AA youths compared with CA youths. Several potential explanations can be given for those ethnicity differences in atherogenic lipids between AA and CA youths.

First, lipoprotein lipase hydrolyzes TG components of circulating TG-rich lipoproteins (TRL) including VLDL and chylomicrons, thereby removing TG from the circulation. ApoC-III is a constituent of VLDL and chylomicrons that is a noncompetitive inhibitor of lipoprotein lipase.[17,18] Previous mice studies have demonstrated that apoC-III level has a direct and dramatic influence on plasma TG levels.[18,19] Similarly, clinical studies reported that apoC-III levels were positively associated with elevated plasma TG levels via delayed clearance of TRLs.[20-22] In a HERITAGE study, Despres et al[23] found that post-heparin-releasable lipoprotein lipase activity was higher while HL activity was lower in AAs versus CAs. In the present study, therefore, elevated apoC-III levels in CA youths compared with AA youths may indicate that TRL clearance from the circulation is less efficient in the former than in the latter, leading to elevated TG levels along with a higher prevalence of relatively smaller LDL particles.

Second, ethnicity differences in those lipoprotein-lipids might be associated with apoE-mediated hepatic uptake of TRL remnants. Plasma apoE increases the clearance rate of TRL remnants through recognition and uptake by hepatic receptors,[24,25] while apoC-III reduces the apoE-mediated hepatic uptake of TRL remnants.[26] In the present study, we found that AA youths had a significantly higher apoE level and a lower apoC-III level than CA youths, suggesting that hepatic uptake of TRL remnants from the circulation might be more efficient in the former than in the latter.

With respect to the ethnicity difference in apoC-III between AA and CA youths, no clear explanation can be given due to the cross-sectional nature of the present study. The apoC-III gene has an insulin-response element on its promoter that negatively affects gene expression.[27] And it has also been suggested that the normal suppression of hepatic apoC-III expression by insulin is diminished in the insulin resistant state, which leads to increased apoC-III expression and TG-rich VLDL particles in the circulation. For example, epidemiologic studies showed that plasma apoC-III was positively associated with hyperinsulinemia,[28] metabolic syndrome,[20] and type 2 diabetes.[21] In the present study, we found that even with higher insulin and HOMA-IR levels, AA youths had a significantly lower apoC-III level than CA youths. In CA, not AA, youths, HOMA-IR along with waist circumference was an independent predictor for plasma apoC-III. Thus it is certainly possible that unlike CA youths, insulin resistance may not lead to increased apoC-III gene expression and its protein levels in AA youths, implying that the functional influence of insulin on the apoC-III gene expression and thereby its protein expression may differ between CAs and AAs. Considering the cross-sectional nature of this study, however, we cannot provide further information on this question.

Finally, several studies have demonstrated the presence of a complex interaction of genetic variants within apoC-III and the apoA-I/C-III/A-IV/A-V cluster with plasma TG concentrations.[29,30] Waterworth et al[29] reported that the -482T allele frequency in the insulin response element of the apoC-III gene promoter was significantly higher in AAs than in CAs. The -482T allele was also associated with higher concentrations of fasting insulin and TG in CAs, while it was related to lower fasting insulin and glucose concentrations in AAs. However, how the apoC-III genotype influences the associations among insulin resistance and plasma apoC-III and thereby lipoprotein-lipids in both AA and CA youths needs to be investigated in a future study.

In summary, the findings of the present study suggest that compared with CA youths, AA youths have a favorable lipoprotein-lipids profile featuring of low TG and high HDLC along with relatively larger LDL particles, and they appear to be associated with relatively low apoC-III and high apoE levels. Considering the fact that AAs have a higher risk for cardiovascular diseases than CAs; however, one important implication of the present findings is that clinical practitioners and health care professionals need to be aware of that lipoprotein-lipids profile alone in AA youths may not appropriately reflect the future risk for cardiovascular diseases including type 2 diabetes mellitus and hypertension.

Funding: This study was supported by a grant from the Korean Research Foundation (KRF-2009-32A-G00046).

Ethical approval: This study was approved by the committee of SungkyunkwanUniversity for Human Subject Research.

Competing interest: No conflicts of interest are declared by the authors.

Contributors: Kang HS conceived and designed the study, analyzed the data, and wrote the first draft. Lee JY and Hong HR contributed to data analysis and interpretation and writing and critical revisions of the manuscript. All the authors reviewed and approved the final version of the manuscript.